Design and generation of a recombinant hepatitis B-based viral vector system for use in cellular receptor studies

grantor: University of TorontoThe hepatitis B virus remains a prevalent global problem despite the availability of recombinant vaccines. One of the greatest challenges facing hepatitis B researchers is the unavailability of an in vitro hepatitis B virus replication system. As a result, initial viral entry steps as well as the cellular receptor for HBV remain undefined. In this study, construction and generation of recombinant hepatitis B viral particles carrying a reporter gene has been attempted. Different immortalized cell lines were tested for their ability to drive hepatitis B promoters. The enhanced green fluorescent protein (EGFP) was placed under control of the different hepatitis B promoter regions and transfected into Chang Liver, Chinese Hamster Ovary (CHO), HeLa, Hepatoblastoma G2 (Hep G2), Human Hepatoma 7 (HuH-7), Ost-7, and Sf9 cells. Only Hep G2 and HuH-7 cells were capable of driving EGFP expression under all HBV promoters. Recombinant baculoviruses carrying replication-defective HBV genomes and HBV packaging genes were generated to deliver HBV DNA into Hep G2 and HuH-7 cells. The ability of these cell lines to secrete replication-defective HBV particles was confirmed using specific PCR reactions and electron microscopy. Further development of this viral system would eventually provide us a useful tool to help elucidate the early stages of the hepatitis B virus replication cycle. This, in turn, will further the development of drugs and therapies for those infected with the hepatitis B virus.M.Sc